Preparation and Comparative Evaluation of Vegetable oil Adjuvaned Foot–and–Mouth Disease Vaccines in Rabbits

Bahar–E–Mustafa^*, Muhammad Arshad, Muhammad Ashraf, Sadia Nasreen, Muhammad Abu Bakr Shabbir, Zeeshan Nawaz

*Corresponding author: baharmsf@yahoo.com

ARTICLE CITATION: Mustafa B, Arshad M, Ashraf M, Nasreen S, Shabbir MAB, Nawaz Z (2014). Preparation and comparative evaluation of vegetable oil adjuvaned foot–and–mouth disease vaccines in rabbits. J. Inf. Mol. Biol. 2 (1): 6 – 10.
Received: 2013–12–31, Revised: 2014–03–01, Accepted: 2014–03–04
The electronic version of this article is the complete one and can be found online at ( http://dx.doi.org/10.14737/jimb.2307-5465/2.1.6.10 ) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

ABSTRACT

The main objective of the present study was to prepare and comparatively evaluate foot–and– mouth disease (serotype O) vaccines having different vegetable oils (peanut, sunflower and olive oil) as adjuvants and determine the possible adverse reactions, they induce. For that purpose, female rabbits (n=30) were divided, randomly into 5 groups, group A, B, C and D were inoculated with aluminium hydroxide, peanut oil, sunflower oil and olive oil respectively (@ 0.4 ml/ml). Booster dose were given (group A to D) 21 days following priming dose. Group E was kept as negative control (given 0.4 ml normal saline). Collection of serum samples were done at the day of vaccination (day 0) and then 7, 14, 21, 28, 42, 56 and 70 days following priming dose of vaccination. Measurement of antibody titers was done by indirect haemagglutination assay. It was found that following the priming dose of vaccination, antibody titers attained by group A were found to be higher as compared to the animals vaccinated with the peanut (group B) and sunflower adjuvanted (group C) vaccines, although this difference was not found to be significant statistically (P>0.05), however, titers of group A, B and C were significantly higher (P<0.05) as compared to the group E. Following the booster dose of immunization, increase in titers of group B, C and D were found to be higher as compared to the group A. However, statistically, GMTs of group A, B, C and D were not found to be significantly different (P>0.05), although they differed significantly (P<0.05) from group E all the times following the booster dose. None of the vaccines induced any localized or systemic reactions (deaths and rise in body temperature) following priming vaccination although they induced localized reactions of mild type following booster dose of the vaccination. The present study recommends the adjuvant action of vegetable oils for foot and mouth vaccine.

INTRODUCTION

Foot and mouth disease (FMD) is an economically important, acute and highly contagious disease of even–toed animal’s like buffalo, cattle, goat, sheep and many other domestic and zoo animals (Bastos et al., 2003). The disease manifests itself clinically by the formation of vesicles on mouth, teats and feet of animals. It results in significant economic losses to dairy farmer in form of decreased production of milk, abortions and weight loss. The disease is a major constraint in the trade of animals (and their products) internationally (James and Rushton, 2002).

The causative agent of the disease is a virus; Apthovirus belonging to the family Picornaviridae (Bablanian and Grubman, 1993).This is a naked, single stranded RNA, positive sense virus having genome size of 8500 base pairs. This virus possess an icosahederal symmetry and four structural proteins (VP1, 2, 3 and 4); among them VP1 is a major immunogenic protein (Cloette et al., 2008).

Proper control of the disease can be achieved by a variety of different methods like controlling the movement of animals and slaughtering of suspected and diseased animals (John, 2002). However, in countries like Pakistan where the disease is endemic in nature, and often appears as outbreaks, it is controlled by the proper vaccination of the animals (Orsel et al., 2007). Currently vaccine is being prepared in the University of veterinary and animal Sciences (UVAS), Lahore and is found to be very effective in terms of post vaccination humoral immune response as well as the protection from clinical disease (Altaf et al., 2012).

Both aluminium hydroxide and saponin (AS) and oils are currently being used as adjuvants of FMD. It is a well-known fact that the oil adjuvants offer a better protection as compared to the AS adjuvanted vaccines. Oil adjuvants provide higher titers of antibodies that tend to persist for a longer duration of time as compared to the AS adjuvanted vaccines (Sadir et al., 1988). Those animals which were immunized with the oil adjuvanted FMD vaccines showed a better protection against homologus virus challenge than those vaccinated with the AS adjuvanted FMD vaccines (Bahnemann et al., 1987). The main disadvantage associated with the use of oil adjuvanted vaccines is that sometimes they result in the severe local reactions such as cyst and granuloma formation. Several factors may be responsible for such reactions, the principle factor involved is the impurities present within oil used as adjuvant. So it is suggested that only those oils should be used which have lower viscosity and the chain length of that hydrocarbon compound should be 12–30 (Bomford, 1977). Hydrocarbons having lower chain length induce more inflammation (Gupta et al., 1993).

So, in view of above mentioned findings, it is better to use that oil as adjuvant of FMD vaccine which not only provides persistent antibody titers against FMD but also does not produce any local adverse reactions. Now a day, a variety of vegetable oils is being used as adjuvants. The major advantage of vegetable oils over mineral oils is that being easily biodegradable compounds, they induce little or no adverse reactions. Moreover, they are easily available, cheap, pure and safe. It has been proved that the vegetable oil adjuvants have potential to enhance the immune response of the host (Gupta et al., 1993). Recently conducted studies showed that corn, rice, canola, soya bean, olive, linseed, rapeseed, peanut, sesame and cashew nuts have potential to stimulate both immune response of the host (Barnett et al., 1996).

In the present study, it was shown that the emulsions made from the peanut oil and sunflower oil were able to enhance the specific humoral response against FMD vaccine.

MATERIALS AND METHODS

FMDV type O strain was procured from WTO– Quality Operations laboratory, University of veterinary & animal Sciences (UVAS), Lahore. Reed and Munch, (1938) method was used to calculate the biological titer of virus and final concentration of the virus was adjusted at 106 units of TCID50 (Altaf et al., 2012). Binary etyhyleneimine (0.01 M) was used as inactivation agent of antigen. Virus inactivation was done at 37oC for 48 hours (Bahnemann, 1975).

Vegetable oils (Peanut, sunflower and olive oil) were used as adjuvants of FMD vaccine and Tween 80 and Span 80 were used as surfactants. Both vegetable oils and surfactants were sterilized by autoclaving at 121oC for 15 minutes. A total of 10% of aqueous phase (Tween–80) and oil phase (Span 80) were added to the vegetable oils (peanut, sunflower and olive oil) as surfactants, so the final hydrsophile lipophile balance (HLB) of the oil adjuvanted FMD vaccine was adjusted at 7.0. One part of inactivated antigen was added into four parts of sterilized vegetable oils (peant, sunflower and olive oil) containing surfactants to make water–in–oil (W/O) emulsion at aqueous to oil ratio of 1:4 (Ezeifeka et al., 2008) (Table 1). Physical parameters of the vaccine like color, viscosity, stability and emulsion type were also studied as described by Stone (1988).

Sterility of all of these vaccines was observed by inoculating each vaccine on different bacteriological media, i.e nutrient agar, MacConkey’s agar, blood agar and Sabouraud’s dextrose agar at 37oC for a period of 24–48 hours (Mahboob et al., 1996). For safety testing, 0.1 ml of each vaccine was inoculated into the tongue of the goats (intradermal), followed by inoculation of 2 ml of vaccine on neck of the animals after 4 days. The animals were monitored for a further 6 days for the presence of any of FMD signs i.e vesicles on mouth, feet and teats (Cloette et al., 2008).

Experimental Model
A total of 30 female rabbits of 6–12 months of age were used. Only those animals were selected which were not vaccinated with FMD vaccine previously. These animals were divided into five groups, each having six animals. These animals were vaccinated with the priming dose of vaccine first and then booster dose was given 21 days after the priming dose, with vaccines containing different adjuvants as follows (Table 2). Group A was used as positive control (given a commercially procured FMD vaccine having AS as adjuant) while group E was kept as negative control.